Multiplexed Isotope-Coded Mass Spectrometry Footprinting Enables Broad Heteroatom-Residue Mapping and Uncovers...

Deciphering novel protein-ligand interaction sites with high structural precision is essential for elucidating protein function and advancing therapeutic development. Mass spectrometry-based protein footprinting offers a powerful means to interrogate binding interfaces; however, broader and more versatile residue labeling is needed to enhance resolution and applicability in complex systems. Here, we developed a footprinting strategy based on Woodward's Reagent K (WRK) targeting heteroatom-containing side chains. The approach is tunable, and it broadly modifies nine nucleophilic residues─Glu, Asp, His, Tyr, Thr, Ser, Lys, Arg, and Cys─representing one of the broadest residue profiles achieved by nonradical footprinting. The WRK scaffold readily accommodates isotope coding, enabling precise, multiplexed quantitative analysis. Applied to two rigid β-sheet proteins, this method accurately maps native ligand-binding sites across a continuum of binding affinities. Remarkably, WRK footprinting delineates a previously uncharacterized ligand-binding pocket on tetrameric transthyretin (TTR) and reveals a noncanonical fibrillogenesis-inhibitory mechanism mediated by a novel ligand identified through high-throughput affinity mass spectrometry screening. This ligand impedes TTR fibril formation by engaging a distinct pocket separate from the canonical T4 binding site. Altogether, these findings position WRK as an effective tool for resolving noncanonical binding sites with therapeutic potential in structure-based drug discovery.